Pluripotent stem cells such as embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) are cells having a capability of differentiating into various cells, and they possess a capability of almost indefinitely proliferating. Recently, particularly in the field of regenerative medicine, there has been a need for development of methods which produce, by use of such pluripotent stem cells as a starting material, tissues and cells applicable to various organs such as stomach, pancreas, liver and intestine. More specifically, as the survival rate of premature babies has rapidly increased due to advancements of neonatal medicine, there is an increased need for a regeneration medicinal technology which is effective to infants with congenital hypoplasia in digestive tracts. Furthermore, since epithelial metaplasia in intestines, or irreversible structural changes in gastrointestinal mucous membranes occur in gastrointestinal malignant tumors, stricture or fibrosis developed after surgeries for said disease, reflux esophagitis, and digestive-tract dysfunction due to tissue destruction that is involved in chronic inflammatory intestinal diseases such as ulcerative colitis and Crohn disease, there has been a need for regenerative-medicine-based therapies therefor. In order to realize regenerative-medicine-based therapies against such digestive system disorders, there has been a urgent need to develop an efficient method of producing intestinal cells by use of pluripotent stem cells as a starting material.
With regard to methods of differentiating embryonic stem cells into endodermal cells, for example, a method in which mesoderm-derived cells are used as feeder cells, and embryonic stem cells are cultured in the presence of said feeder cells to thereby induce differentiation of them into endodermal cells (see WO2006/126574). The patent document WO2006/126574 describes induction of differentiation thereof into mature cells of endoderm-derived organs such as liver, lung, and small intestine, but the disclosed method cannot efficiently differentiate the cells into various matured intestinal cells.
Moreover, techniques have been established, in which ES cells are culture on a monolayer of M15 cells in vitro to thereby induce the ES cells sequentially into the mesendoderm, the definitive endoderm, and, finally, various organs derived from the regional-specific definitive endoderm, as they mimic in vivo induction of early embryos [see Shiraki, N., Umeda, K., Sakashita, N., Takeya, M., Kume, K. and Kume, S. (2008). Differentiation of mouse and human embryonic stem cells into hepatic lineages. Genes Cells 13, 731-46; and Shiraki, N., Yoshida, T., Araki, K., Umezawa, A., Higuchi, Y., Goto, H., Kume, K. and Kume, S. (2008b). Guided differentiation of embryonic stem cells into Pdx1-expressing regional-specific definitive endoderm. Stem Cells 26, 874-851. It has been confirmed that these techniques have succeeded in inducing differentiation of the ES cells into hepatic cells, pulmonary cells, pancreatic cells and the like. Particularly, the document of Shiraki et al (2008b) describes that Cdx2-expressing intestinal precursor cells also were generated besides hepatic, pulmonary, and pancreatic cells. However, it is difficult to produce various types of more mature intestinal cells massively and effectively by use of these conventional arts.
As described above, techniques for inducing differentiation of pluripotent stem cells into various types of mature intestinal cells massively and effectively still remain to be developed. At present, any efficient methods of producing intestinal cells by use of pluripotent stem cells as a starting material do not exist.